U.S. patent application number 10/758793 was filed with the patent office on 2004-10-28 for red to near-infrared photobiomodulation treatment of the visual system in visual system disease or injury.
Invention is credited to Eells, Janis T., Whelan, Harry T., Wong-Riley, Margaret T. T..
Application Number | 20040215293 10/758793 |
Document ID | / |
Family ID | 33302888 |
Filed Date | 2004-10-28 |
United States Patent
Application |
20040215293 |
Kind Code |
A1 |
Eells, Janis T. ; et
al. |
October 28, 2004 |
Red to near-infrared photobiomodulation treatment of the visual
system in visual system disease or injury
Abstract
A method of treating visual system disease is disclosed. One
embodiment comprises the steps of (a) exposing a component of a
patient's visual system to light treatment, wherein the light
treatment is characterized by wavelength of between 630-1000 nm and
power intensity between 10-90 mW/cm.sup.2 for a time of 1-3
minutes, and (b) observing restoration of visual system
function.
Inventors: |
Eells, Janis T.; (Madison,
WI) ; Wong-Riley, Margaret T. T.; (Brookfield,
WI) ; Whelan, Harry T.; (Whitefish Bay, WI) |
Correspondence
Address: |
QUARLES & BRADY LLP
411 E. WISCONSIN AVENUE
SUITE 2040
MILWAUKEE
WI
53202-4497
US
|
Family ID: |
33302888 |
Appl. No.: |
10/758793 |
Filed: |
January 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60440816 |
Jan 17, 2003 |
|
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Current U.S.
Class: |
607/89 |
Current CPC
Class: |
A61N 5/0613 20130101;
A61N 2005/0659 20130101; A61N 2005/0652 20130101 |
Class at
Publication: |
607/089 |
International
Class: |
A61N 001/00 |
Goverment Interests
[0002] This invention was made with United States government
support awarded by the following agencies: Defense Advanced
Research Projects Agency Grant DARPA N66001-01-1-8969 and
N66001-03-1-8906, National Institute of Environmental Health
Sciences Grant ES06648, National Eye Institute Core Grant
P30-EY01931, National Eye Institute Grants EY11396 and EY05439. The
United States has certain rights in this invention.
Claims
We claim:
1. A method of treating visual system disease or injury, comprising
the steps of a) exposing a component of a patient's visual system
to light treatment, wherein the light treatment is characterized by
wavelength between 630-1000 nm and power intensity between 10-90
mW/cm.sup.2 for a time of 1-3 minutes, and b) observing restoration
or protection of visual function.
2. The method of claim 1 wherein the wavelength is selected from
the group consisting of 670 nm, 830 nm and 880 nm.
3. The method of claim 1 wherein the wavelength is between 670-900
nm.
4. The method of claim 1 wherein the light treatment is
characterized by an energy density of between 0.5-20
J/cm.sup.2.
5. The method of claim 4 when the energy density is between 2-10
J/cm.sup.2.
6. The method of claim 1 wherein the patient is exposed to light
treatment multiple times.
7. The method of claim 6 wherein the exposure is at least 3
times.
8. The method of claim 1 wherein the patient is exposed to light
treatment intervals of 24 hours.
9. The method of claim 1 wherein the treatments are administered
2-3 times per day.
10. The method of claim 1 wherein the component of the visual
system comprises the patient's retina.
11. The method of claim 1 wherein the component of the visual
system is selected from the group consisting of cornea and optic
nerve.
12. The method of claim 1 herein the retinal function is
evaluated.
13. The method of claim 1 wherein the light is supplied by an LED
device.
14. The method of claim 1 wherein the power intensity is between
25-50 mW/ cm.sup.2.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. provisional
application 60/440,816, filed Jan. 17, 2003, incorporated by
reference herein.
BACKGROUND OF THE INVENTION
[0003] Decrements in mitochondrial function have been postulated to
be involved in the pathogenesis of numerous retinal and optic nerve
diseases, including age-related macular degeneration, diabetic
retinopathy, and Leber's hereditary optic neuropathy (J. F. Rizzo,
Neurology 45:11-16, 1995; M. J. Baron, et al., Invest. Ophthalmol.
Visual Sci. 42:3016-3022, 2001; V. Carelli, et al., Neurochem. Int.
40:573-584, 2002). Decrements in mitochondrial function have also
been postulated to be involved in the pathogenesis in methanol
intoxication (M. M. Hayreh, et al., Neurotoxicity of the Visual
System, eds. Merigan, W. & Weiss, B. (Raven, New York), pp.
35-53, 1980; G. Martin-Amat, et al., Arch. Ophthalmol.
95:1847-1850, 1977; M. T. Seme, et al., J. Pharmacol. Exp. Ther.
289:361-370, 1999; M. T. Seme, et al., Invest. Ophthalmol. Visual
Sci. 42:834-841, 2001). Methanol intoxication produces toxic injury
to the retina and optic nerve, frequently resulting in blindness. A
toxic exposure to methanol typically results in the development of
formic academia, metabolic acidosis, visual toxicity, coma, and, in
extreme cases, death (J. T. Eells, Browning's Toxicity and
Metabolism of Industrial Solvents: Alcohols and Esters, eds.
Thurman, T. G. Kaufmann, F. C. (Elsevier, Amsterdam), Vol. IV, pp.
3-15, 1992; R. Kavet and K. Nauss, Crit. Rev. Toxicol. 21:21-50,
1990). Visual disturbances generally develop between 18 and 48
hours after methanol ingestion and range from misty or blurred
vision to complete blindness. Both acute and chronic methanol
exposure have been shown to produce retinal dysfunction and optic
nerve damage clinically (J. T. Eells, supra, 1992; R. Kavet and K.
Nauss, supra, 1990; J. Sharpe, et al., Neurology 32:1093-1100,
1982) and in experimental animal models (S. O. Ingemansson, Acta
Ophthalmol. 158(Supp):5-12, 1983; J. T. Eells, et al.,
Neurotoxicology 21:321-330, 2000; T. G. Murray, et al., Arch.
Ophthalmol. 109:1012-1016, 1991; E. W. Lee, et al., Toxicol. Appl.
Pharmacol. 128:199-206, 1994).
[0004] Formic acid is the toxic metabolite responsible for the
retinal and optic nerve toxicity produced in methanol intoxication
(M. M. Hayreh, et al., supra, 1980; G. Martin-Amat, et al., supra
1977; M. T. Seme, et al., supra, 1999; M. T. Seme, supra, 2001; G.
Martin-Amat, et al., Toxicol. Appl. Pharmacol. 45:201-208, 1978).
Formic acid is a mitochondrial toxin that inhibits cytochrome c
oxidase, the terminal enzyme of the mitochondrial electron
transport chain of all eukaryotes (P. Nicholls, Biochem. Biophys.
Res. Commun. 67:610-616, 1975; P. Nicholls, Biochim. Biophys.
Acta430:13-29, 1976). Cytochrome oxidase is an important
energy-generating enzyme critical for the proper functioning of
almost all cells, especially those of highly oxidative organs,
including the retina and brain (M. T. T. Wong-Riley, Trends
Neurosci. 12:94-101, 1989). Previous studies in our laboratory have
established a rodent model of methanol-induced visual toxicity and
documented formate-induced mitochondrial dysfunction and retinal
photoreceptor toxicity in this animal model (M. T. Seme, et al.,
supra, 1999; M. T. Seme, et al., supra, 2001; J. T. Eells, et al.,
supra, 2000; T. G. Murray, et al., supra, 1991).
[0005] Photobiomodulation by light in the red to near-IR range
(630-1,000 nm) using low-energy lasers or light-emitting diode
(LED) arrays has been shown to accelerate wound healing, improve
recovery from ischemic injury in the heart, and attenuate
degeneration in the injured optic nerve (H. T. Whelan, et al., J.
Clin. Laser Med. Surg. 19:305-314, 2001; U. Oron, et al., Lasers
Surg. Med. 28:204-211, 2001; E. M. Assa, et al., Brain Res.
476:205-212, 1989; M. J. Conlan, et al., J. Clin. Periodont.
23:492-496, 1996; W. Yu, et al., Lasers Surg. Med. 20:56-63, 1997;
A. P. Sommer, et al., J. Clin. Laser Med. Surg. 19:27-33, 2001). At
the cellular level, photoirradiation at low fluences can generate
significant biological effects, including cellular proliferation,
collagen synthesis, and the release of growth factors from cells
(M. J. Conlan, et al., supra, 1996; T. Karu, J. Photochem.
Photobiol. 49:1-17, 1999; M. C. P. Leung, et al., Lasers Surq. Med.
31:283-288, 2002). Our previous studies have demonstrated that LED
photoirradiation at 670 nm (4 J/cm.sup.2) stimulates cellular
proliferation in cultured cells and significantly improves wound
healing in genetically diabetic mice (H. T. Whelan, et al., supra,
2001; A. P. Sommer, et al., supra, 2001). Despite its widespread
clinical application, the mechanisms responsible for the beneficial
actions of photobiomodulation have not been elucidated.
Mitochondrial cytochromes have been postulated as photoacceptors
for red to near-IR light energy and reactive oxygen species have
been advanced as potential mediators of the biological effects of
this light (Karu, supra, 1999; N. Grossman, et al., Lasers Surg
Med. 22:212-218, 1998).
BRIEF SUMMARY OF THE INVENTION
[0006] In one embodiment, the present invention is a method of
treating disease or injury of the visual system, comprising the
steps of (a) exposing a component of a patient's visual system to
light treatment, wherein the light treatment is characterized by
wavelength between 630-1000 nm and power intensity between 10-90
mW/cm.sup.2 for a time of 1-3 minutes, and (b) observing
restoration or protection of visual system function. Preferably,
the wavelength is selected from the group consisting of 670 nm, 830
nm and 880 nm.
[0007] In one embodiment of the invention, the light treatment is
characterized by an energy density of between 0.5-20 J/cm.sup.2. In
a preferred embodiment, the energy density is between 2-10
J/cm.sup.2.
[0008] Preferably, the patient is exposed to light treatment
multiple times and is exposed to light treatment intervals of 24
hours.
[0009] In a preferred form of the invention, the component of the
visual system comprises the patient's retina or is selected from
the group consisting of cornea and optic nerve.
[0010] Other features, objects or advantages of the present
invention will become apparent to one of skill in the art after
examination of the specification, claims and drawings.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0011] FIG. 1 is a graph of wavelength versus relative absorbance,
content or activity.
[0012] FIG. 2 is a graph of rod and M-cone ERG amplitude (.mu.V)
versus log relative retinal illumination.
[0013] FIG. 3 is a graph of UV-cone ERG amplitude (.mu.V) versus
log relative retinal illumination.
[0014] FIG. 4(A-D) is a set of micrographs illustrating outer
retinal morphology in representative untreated control (FIG. 4A),
LED control (FIG. 4B), methanol-intoxicated (FIG. 4C), and
LED-treated, methanol-intoxicated (FIG. 4D) retinas.
[0015] FIG. 5(A-D) is a set of electron micrographs of the rod
inner segment region in representative untreated control (FIG. 5A)
LED control, (FIG. 5B) methanol-intoxicated, (FIG. 5C) and
LED-treated, methanol-intoxicated (FIG. 5D) rats.
[0016] FIG. 6(A-B) is a set of graphs. FIG. 6A is a graph of rod
and M-cone ERG amplitude versus log relative retinal illumination.
FIG. 6B is a graph of ERG amplitude versus log relative retinal
illumination.
[0017] FIGS. 7A and B describes NIR LED treatment as improving
healing following laser-induced retinal injury. FIG. 7A is a set of
micrographs of a laser grid at 15 minutes and 1 month post-laser
treatment in both LED-treated and non-LED-treated tissue. FIG. 7B
is a bar graph of severity of burn (spot persistence) versus
treatment.
[0018] FIG. 8 describes NIR LED treatment as improving visual
function. FIG. 8A is a set of micrographs of lateral geniculate
nuclei of monkeys with monocular central retinal laser injury both
treated and not treated with NIR-LED. FIG. 8B is a bar graph of
untreated and LED-treated subjects versus percentage of
metabolically active neurons in layer 6 of LGN.
[0019] FIG. 9 describes NIR-LED treatment as improving retinal
function. FIG. 9 is a graph of the multifocal ERG response in
nanovolts versus pre-laser, post-laser, four day post-laser and
eleven day post-laser treatment for LED-treated and non-treated
tissue.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Low energy photon irradiation by light in the far red to
near infrared spectral (range 630-1000 nm) using low energy lasers
or light emitting diode arrays has been found to modulate various
biological processes in cell culture and animal models [(M. J.
Conlan, et al., J. Clin. Periodont. 23:492-496, 1996; W. Yu, et
al., Lasers Surg. Med. 20:56-63, 1997; A. P. Sommer, et al., J.
Clin. Laser Med. Surg. 19:27-33, 2001; T. Karu, J. Photochem.
Photobiol. 49:1-17, 1999)]. As described above, this phenomenon of
photobiomodulation has been applied clinically in the treatment
soft tissue injuries and to accelerate wound healing [(H. T.
Whelan, et al., J. Clin. Laser Med. Surg. 19:305-314, 2001; U.
Oron, et al., Lasers Surg. Med. 28:204-211, 2001; E. M. Assa, et
al., Brain Res. 476:205-212, 1989; M. J. Conlan, et al., supra,
1996)]. The mechanism of photobiomodulation by red to near infrared
light at the cellular level has been ascribed to the activation of
mitochondrial respiratory chain components resulting in initiation
of a signaling cascade which promotes cellular proliferation and
cytoprotection.
[0021] The therapeutic effects of red to near infrared light may
result, in part, from the stimulation of cellular events associated
with increases in cytochrome c oxidase activity. In support of this
theory, we have demonstrated in primary neuronal cells that LED
photobiomodulation (670 nm at 4 J/cm.sup.2) reverses the reduction
in cytochrome oxidase activity produced by the blockade of
voltage-dependent sodium channel function by tetrodotoxin (M. T. T.
Wong-Riley, et al., NeurReport 12:3033-3037, 2001). In addition, we
have shown that the action spectrum for LED stimuation of
cytochrome oxidase activity and cellular ATP content parallels the
absorption spectrum for cytochrome oxidase. (FIG. 1). The
structured nature of the action spectrum is strong evidence that
cytochrome oxidase is a primary photoacceptor molecule for light in
the far red to near infrared region of the spectrum. Our recent
work has provided evidence for the therapeutic benefit of
photobiomodulation in the survival and functional recovery of the
retina and optic nerve in vivo after acute injury by the
mitochondrial toxin, formic acid generated in the course of
methanol intoxication. (Eells, et al., Proc. Natl. Acad. Sci.
100(6):3439-3441, incorporated by reference herein.) In addition,
we have provided data below indicating that 670 nm LED treatment
promotes retinal healing and improved visual function following
high intensity laser-induced retinal injury in adult non-human
primates.
[0022] These findings provide a link between the actions of red to
near infrared light on mitochondrial oxidative metabolism in vitro
and ocular injury in vivo. Importantly, the results of these
studies and others suggested to us that photobiomodulation with red
to near infrared light augments recovery pathways promoting
neuronal viability and restoring neuronal function following ocular
injury. There was no evidence of damage to the retina or optic
nerve following 670 nm LED treatment. Based on these findings, we
suggest that photobiomodulation represents a non-invasive and
innovative therapeutic approach for the treatment of ocular injury
and acute and chronic ocular disease.
[0023] In one broad aspect, the present invention is a method of
treating ocular disease comprising the steps of exposing a
component of a patient's visual system to light treatment wherein
the light treatment is characterized by wavelength between 630-1000
nm and power intensity between 25-50 mW/cm.sup.2 for a time of 1-3
minutes (equivalent to an energy density of 2-10 J/cm.sup.2) and
observing restoration or protection of visual function. The
sections below further describe and characterize the present
invention.
[0024] Suitable Led
[0025] In a preferred form of the present invention, the
therapeutic photobiomodulation is a achieved using an light
emitting diode as the source of red to near infrared light unlike
the prior art which utilized light generated by low energy laser.
Lasers have limitations in beam width, wavelength capabilities, and
size of the injury or tissue that can be treated. In addition, heat
generated from laser light can damage biological tissue, and the
concentrated beam of laser light may accidentally damage the eye.
Light-emitting diode (LED) arrays were developed for the National
Aeronautics and Space Administration manned space flight
experiments. In comparison with lasers, LED technology generates
negligible amounts of heat, is clinically proven to be safe, and
has achieved non-significant risk status for human trials by the
U.S. Food and Drug Administration.
[0026] Preferably, the present inventions utilizes a noncoherent
light source capable of irradiating the entire retina and optic
system with monochromatic light in the far-red to near-infrared
region of the spectrum. An example of a commercially available,
preferable LED source is the ISO 9001 LED (QUANTUM SPECTRALIFE)
array obtainable from Quantum Devices (Barneveld, Wis.).
[0027] Light in the far-red to near infrared region of the spectrum
is known to penetrate nearly 20 cm into irradiated tissue. A
preferable device is composed of a monolithic array of hybrid
GaAIAs light emitting diodes designed to emit diffused
monochromatic light. Preferable LED chips have been fabricated to
emit specific peak wavelengths (between 650-940 nm) of photon
energy and the system has been designed to deliver high intensity
light energy to an isolated area of exposure without heat.
[0028] Preferably, the LEDs are shielded by a glass barrier and are
unlensed, allowing for even dispersion of light over a 180.degree.
viewing angle allowing each LED chip to act as a point source
offering a high degree of illumination uniformity. The surface
energy (photon flux or power intensity) delivered by the LED units
is between 25-50 mW/cm.sup.2. LED units that we have worked with
produce monochromatic light at wavelengths in the far red (670 nm)
to near infrared (700-900 nm) region of the spectrum. The LED
arrays can be custom fabricated from GaAIAs (galinium aluminum
arsinate) diodes to produce light with peak wavelengths between 650
nm and 940 nm. The devices that we have employed have peak
wavelengths of 670, 728, 830 and 880 nm with bandwidths of 25-35 nm
at 50% power. LED units and low energy lasers could be constructed
that emit at other peak wavelengths in the range of 630-1000
nm.
[0029] Peak wavelengths of 670 nm, 830 nm, and 880 nm with
bandwidths of 25-30 nm have been used with success in experimental
and clinical studies. Both benchtop LED units (bandwidth 25-30 cm
at 50% power) with 8.times.10 cm rectangular arrays and portable
NIR-LED units with 5 cm diameter circular arrays obtained from
Quantum Devices, Inc. (Barneveld, Wis.) have been used in our
experimental and clinical studies. In our experimental and clinical
studies of NIR-LED treatment of ocular injury or disease, 670 nm
LED irradiation was administered at a power intensity between 25-50
mW/cm.sup.2 between 1-3 minutes to produce an energy density of 4
J/cm.sup.2.
[0030] In a preferred embodiment of the present invention, 670 nm
LED irradiation administered at a power intensity between 25-50
mW/cm.sup.2 between 1-3 minutes produces an energy density of 4
J/cm.sup.2 and promotes retinal healing and improves visual
function. It is likely that other NIR wavelengths (830, 880 nm)
will also promote retinal healing and protect against retinal
injury, but we have no animal model evidence for this at this
time.
[0031] The LED unit is typically positioned at a distance of
approximately 2.5 cm from the eye in each case. We envision that
one would wish to position the light source at between 0.5 cm and
4.0 cm from the eye or 0.5-1.0 cm from the top, side or back of the
head for irradiation of other components (optic nerve, lateral
geniculate nucleus, superior colliuculus, etc.) of the visual
system. As described below, the position from the target factors
into the entire energy density calculation.
[0032] Optimal Treatment Protocols
[0033] There are four important treatment parameters variables in
the therapy of the present invention: (1) Energy density or
fluence, (2) light wavelength (3) number of treatments and (4) the
treatment interval.
[0034] (1) Energy density or fluence is the product of LED power
intensity and duration of irradiation and is expressed as Joules
per square centimeter (J/cm.sup.2). For effective NIR-light therapy
of the present invention, the energy density cannot be too low,
otherwise there will be no biological effect. Energy density should
also not be too great or it might produce adverse effects. Prior
studies have suggested that biostimulation occurs at energy
densities between 0.5 and 20 J/cm.sup.2, whereas energy densities
above 20 J/cm.sup.2 exert bioinihibitory effects. Preferable energy
density of the present invention is between 0.5-20 J/cm.sup.2, most
preferably between 2-10 J/cm.sup.2. This range is based on evidence
which documents that exposure to near-infrared light at energy
densities (fluence) between 2-10 J/cm.sup.2 promotes cellular
energy metabolism, cell division and wound healing, protects
against toxin-induced retinal damage and promotes healing and
improved visual function following high intensity laser-induced
retinal injury.
[0035] (2) With respect to LED wavelength, the majority of our
studies have been conducted using 670 nm LED light and there is
substantial evidence that NIR- LED treatment at 670 nm is
beneficial for the treatment of ocular toxicity, retinal laser
injury and retinal disease. NIR-LED light at wavelengths
corresponding to the absorption peaks of the copper centers in the
cytochrome oxidase molecule (670 nm, 830 nm and 880 nm) have been
shown to be effective in promoting the recovery of cytochrome
oxidase activity and energy metabolism in cultured primary neurons.
These three wavelengths are preferable wavelengths in the present
invention. (M. Wong-Riley, et al., EPEC Conference, European
Bioenergetics Conference, 2002)
[0036] Band width can vary depending on technology and type of
light source used. Although LED arrays are preferred in the present
invention, the invention includes any far-red to near infrared
light source (low energy lasers and LED arrays) which can produce
energy densities between 0.5-20 J/cm.sup.2. Preferable bandwidth
for low energy laser light sources would be 4 nm and preferable
band width for LEDs would be 25-50 nm. The entire preferred range
would be 4-50 nm.
[0037] (3) In acute ocular injury situations, the optimal time for
initial NIR-LED treatment should be within 24 hours of injury, if
possible, based on our studies of acute ocular injury in rodent and
primate models. However, if treatment is not feasible within the
first 24 hours, it should be initiated as soon as possible.
Molecular studies document upregulation of genes encoding energy
producing and antioxidant proteins within 24 hours of NIR-LED
treatment.
[0038] In summary, a preferred form of the present invention uses
near infrared wavelengths of 630-1000, most preferably, 670-900 nm
(bandwidth of 25-35 nm) with an energy density exposure of 0.5-20
J/cm.sup.2, most preferably 2-10 J/cm.sup.2, to produce
photobiomodulation. This is accomplished by applying a target dose
of 10-90, preferably 25-50 mW/cm.sup.2 LED generated light for the
time required to produce that energy density. Time requirements are
calculated as: Power-intensity (mW/cm.sup.2).times.Time
(seconds).div.1,000=Energy Density (J/cm.sup.2).
[0039] (4) Treatment intervals of 24 hours have been shown to be
beneficial for ocular injuries. Other studies have documented
efficacy with treatments administered 2-3 times per day. It is
likely that treatment spaced 2-3 days apart may also be effective.
For chronic diseases, NIR-LED treatments administered at weekly
intervals may be beneficial.
[0040] We suggest a treatment regime as described below, at 5, 25
and 50 hours. We suggest that the treatment be within 1-3 minutes
of duration to provide the appropriate energy density.
[0041] (5) Number of treatments: We have demonstrated success in
treatment of acute ocular injuries with as few as 2, preferably 3
or more, treatments and as many as 21 treatments administered at 1
day intervals. For the treatment of chronic diseases, NIR-LED
treatments may be administered indefinitely.
[0042] Suitable Treatment Methods
[0043] In a preferred embodiment of the present invention, one
would expose any component of a patient's visual system to the
therapeutic effects of the light treatment described above. By
"visual system" we mean to include the cornea, iris, lens, retina,
optic nerve, optic chiasm, lateral geniculate nucleus, superior
colliculus, pretectal nucleic, the accessory optic system, the
oculomotor system, pulvinar, optic radiations, visual cortex, and
associational visual cortical areas.
[0044] Exposure of the visual system may occur by treating with
light directed into the eyes, thus irradiating the cornea, lens,
iris, retina and optic nerve head. Alternatively, the device can be
oriented so that the light is directed through the back of the
head, thus irradiating the visual cortex or through the sides or
top of the head thus irradiating the other components of the visual
system.
[0045] One would wish to observe restoration or protection of
visual function as measured in any conventional way that assesses
visual function.
[0046] Therapeutic endpoints for treatment of corneal abrasion
would include absence of fluorescein staining of the cornea. For
retinal injury or disease, therapeutic endpoint measurement would
include: (1) Fundoscopy or fundus photography which is an
assessment of the appearance of the fundus or back of the eye,
(note that the retina and optic nerve are observed by using special
lenses); (2) Optical coherence tomography which measures the
thickness (cross sectional architecture) of the retina; (3) Flash,
flicker or multifocal electroretinogram recordings which measure
the electrical response of the rod and cone photoreceptors in the
retina to a light stimulus; (4) The visual evoked cortical
potentials which access the integrity of the retino-geniculo
striate pathway by measuring the electrical response of the visual
area of the brain recorded from scalp electrodes to color vision
testing; and (5) Visual acuity assessment using optotype
(Snellen-style) eye charts. One would expect to see improvement or
protection of the retina as measured by the methods described
above.
[0047] For the optic nerve, therapeutic endpoint measurement would
include the measurement of the visual evoked cortical potential
from regions of the LGN or superior colliculus, to which the optic
nerves project and the Pupillary Light Reflex test, which tests the
integrity of the optic nerve (cranial nerve 2) and the oculomotor
nerve (cranial nerve 3).
[0048] Therapeutic endpoints for improvement of visual function
(measuring LED improvement of disease or injury to other components
of the visual system--optic nerve, LGN visual pathways, etc.)
preferably involves the use of a battery of tests which serve as
standardized assessments for evaluation of the visual functions
important in ensuring that visual perceptual processing is
accurately completed. These include assessment of visual acuity
(distant and reading), contrast sensitivity function, visual field,
oculomotor function visual attention and scanning.
[0049] More detailed descriptions of retinal and visual function
tests include:
[0050] 1. Kinetic (Goldmann) perimetry ("Perimetry" is the
quantitative testing of the side vision).
[0051] 2. Automated (computerized) perimetry. In this test, spots
of light are automatically projected into predetermined areas of
the visual field. The test continues until the dimmest light is
found that can be seen in each area of the side vision. These
visual field tests provide important information.
[0052] 3. Critical Flicker Fusion Frequency (CFF). Patients view a
flickering light to test the ability of the optic nerve to conduct
impulses with uniform speed. This test has proven to be very useful
in identifying visual loss due to optic nerve damage.
[0053] 4. Infra-red video pupillography. This is a way of seeing
the pupils clearly in the dark so that a more certain diagnosis can
be made. We also use it to transilluminate the iris to identify
local iris causes for pupillary abnormalities.
[0054] 5. Electroretinography. A regular ERG (eletroretinogram)
records the electrical activity of the whole retina in response to
light and helps to tell us if the rods and cones of the retina are
firing in the correct way.
[0055] 6. The Multi-focal ERG (MERG) does about a hundred ERGs at
once by illuminating various little bits of the retina
sequentially. It uses a computer to sort out the dizzying torrent
of information and then it presents a map of the sensitivity of
various parts of the retina, based on the electrical activity (in
response to light) of all those different regions. If this map
matches the map from perimetry, then the problem is in the retina
and not in the optic nerve or brain.
[0056] 7. Multi-focal Visual-Evoked Potentials (MVEP). Using a MERG
stimulus, information can be picked up from the scalp that tells us
if the visual pathways in the brain are damaged.
[0057] 8. Computer controlled infra-red sensitive pupillography.
This method is used to monitor pupillary movements in response to
different types of light in order to quantify how much damage there
might be in the visual system.
[0058] 9. Computer controlled "Pupil" Perimetrv. This method uses
the pupil movement in response to small lights presented in the
field of vision as an objective indicator of how well the eye sees
the light.
[0059] 10. Computer recording of eye movements. This instrument can
be used for monitoring pupil movements--but it also has the
capacity to record the small movements of both eyes at the same
time to see if they are tracking together and have normal movements
in different directions of gaze.
[0060] 11. Optical Coherence Tomography (OCT). This is a new device
that looks at the retina at the back of the eye and measures the
thickness of the layer of nerves coming from all quadrants of the
retina and leading into the optic nerve. This nerve fiber layer may
be thickened, thinned or normal, depending on the nature of the
disease affecting the optic nerve.
[0061] 12. Ishihara Color Vision Test Cards. Used for color vision
evaluation. A test chart on color dots that appear as identifiable
numbers or patterns to individuals who have various types of color
vision deficits.
[0062] The retina is a complex sensory organ composed of different
cell types arranged in distinct layers. The term "retinal function"
will be used to refer to (1) activation of these layers by a light
stimulus and (2) the processes required for maintenance of the
cell. Different diseases may affect the retinal layers or cell
types in a selective fashion. Congenital stationary night blindness
affects transmission of visual signals in the rod-mediated visual
pathway whereas achromatopsia affects only the cone pathway. Other
diseases may affect both photoreceptor types in a defined location
on the retina. Examples are the macular dystrophies, such as
Stargardt's and age-related macular degeneration. Other diseases,
such as glaucoma or optic neuropathy appear to affect primarily the
ganglion cells, located on the surface of the inner retina.
[0063] Assessment of the efficacy of a therapeutic intervention in
one of these retinal diseases therefore depends on the specific
disorder. Congenital stationary nightblindness would be best
assessed by the full-field electroretinogram in a patient that has
been adapted to darkness for about 30 minutes. Conversely
achromatopsia, absent cone function, is best assessed by a
full-field electroretinogram under light-adapted conditions and
with a rapidly flickering flash stimulus that isolates cone
function. Diseases of the macula are evident in the multifocal ERG,
but not the full-field. This is due to the fact the macula, with
several hundred thousand photoreceptors makes a very small
contribution to the full-field ERG signal, which is the sum of 12
million or more photoreceptors. For this reason, assessment of the
therapeutic efficacy of an intervention to treat Stargardt's
disease or age-related macular degeneration, would be best
accomplished by the multifocal ERG. Neither full-field ERGs nor
multifocal ERGs contain a significant contribution from the
ganglion cell layer. Assessment of interventions to affect the
progression of glaucoma or Leber's hereditary optic neuropathy thus
use the visually-evoked cortical potential because the visual
cortical response is wholly-dependent on ganglion cell function and
because the ERG is not affected in these diseases.
[0064] The summary, there are a number of different tests used in
clinical ophthalmology that are designed to objectively measure the
function of the retina. The retina must perform a number of jobs in
order to convert a quantum of light entering the eye into an action
potential in the visual cortex. The activation of the retinal
layers by light results in the generation of electric fields in
various levels of the visual system that can be recorded
non-invasively. In theory, the NIR therapy could be beneficial in a
wide range of diseases since it appears to affect basic cellular
responses to insult such as ATP production and apoptosis. Thus
there would be no one test that would be appropriate to assessing
all the diseases that might benefit for NIR therapy.
[0065] For further assessment information, one may wish to consult
American Optometric Association (AOA), Comprehensive adult eye and
vision examination: Reference Guide for Clinicians. St. Louis
(Mo.): American Optometric Association (AOA); 1994; Clinical
Ophthalmology: A Systematic Approach by Jack J. Kanski, et al.,
Butterworth-Heinemann Medical; 5th edition (Jun. 2, 2003fe); A
Textbook of Clinical Ophthalmology, 2nd Edition; and A Practical
Guide to Disorders of the Eyes and Their Management by Ronald Pitts
Crick (King's College Hospital, London) and Peng Tee Khaw
(Moorfields Eye Hospital, London); and Noninvasive Diagnostic
Techniques in Ophthalmology Barry R. Masters (Editor), ISBN:
0387969926, Pub. Date: August 1990 Publisher: Springer-Verlag New
York, Incorporated.
[0066] Treatment Candidates
[0067] Numerous ocular diseases and injuries are likely to benefit
from NIR-LED therapy. These include: 1) Acute ocular injuries to
the cornea, retina and optic nerve, such as corneal abrasions,
acute retinal ischemia, retinal detachment, light or laser induced
retinal injuries. 2) Intoxications affecting the visual system
following exposure to, or ingestion of, environmental toxins (e.g.
methanol, pesticides) and drugs (e.g. ethambutal). 3) Chronic
retinal and optic nerve diseases including, but not limited to,
glaucoma, age-related macular degeneration, diabetic retinopathy,
Leber's hereditary optic neuropathy, and other mitochondrial
diseases with ocular manifestations. 4) Retinopathies and optic
neuropathies resulting from nutritional deficiencies (e.g. folate,
vitamin B.sub.12). 5) Lesions of any centers in the visual system,
including the optic nerve, optic chiasm, optic radiation, dorsal
lateral geniculate nucleus, superior colliculus, and the visual
cortex. Lesions could be induced by accident, trauma, hemorrhage,
blood clot, ischemia, tumor, inflammation, infection or genetic
defects.
EXAMPLES
Example 1
LED Treatment Protects the Rat Retina from Histopathic Changes
Induced by Methanol-Derived Formate.
[0068] In General
[0069] We hypothesize that the therapeutic effects of red to
near-IR light result, in part, from the stimulation of cellular
events associated with increases in cytochrome c oxidase activity.
In support of this hypothesis, we have recently demonstrated in
primary neuronal cells that LED photobiomodulation (670 nm at 4
J/cm.sup.2) reverses the reduction in cytochrome oxidase activity
produced by the blockade of voltage-dependent sodium channel
function by tetrodotoxin (M. T. T. Wong-Riley, et al., NeuroReport
12:3033-3037, 2001). The present studies extended these
investigations to an in vivo system to determine whether 670-nm LED
treatment would improve retinal function in an animal model of
methanol-induced mitochondrial dysfunction.
[0070] Using the electroretinogram (ERG) as a sensitive indicator
of retinal function, we demonstrated that three brief (2 minutes,
24 seconds) 670-nm LED treatments 4 J/cm.sup.2) delivered 5, 25,
and 50 hours after the initial dose of methanol attenuated the
retinotoxic effects of methanol-derived formate. Our studies
demonstrate a significant recovery of rod- and M-cone mediated
retinal function as well as a significant recovery of UV-cone
mediated function in LED-treated rats. We further show that LED
treatment protected the retina from methanol-induced
histopathology. The present study provides evidence that 670 nm LED
treatment promotes the recovery of retinal function and protects
the retina against the cytotoxic actions of the mitochondrial
toxin, formic acid. Our findings are consistent with hypothesis
that LED photobiomodulation at 670 nm improves mitochondrial
respiratory chain function and promotes cellular survival in vivo.
They also suggest that photobiomodulation may enhance recovery from
retinal injury and from other ocular diseases in which
mitochondrial dysfunction is postulated to play a role.
[0071] Methods
[0072] Materials. LED arrays (8.times.10 cm) were obtained from
Quantum Devices (Barneveld, Wis.). Methanol (HPLC grade) obtained
from Sigma was diluted in sterile saline and administered as a 25%
(wt/vol) solution. Thiobutabarbitol sodium salt (Inactin) was
purchased from Research Biochemicals (Natick, Mass.). Atrpine
sulfate was obtained from AmVet Pharmaceuticals (Fort Collins,
Colo.). Hydroxypropyl methylcellulose (2.5%) drops were acquired
from IOLAB Pharmaceuticals, Claremont, Calif. Atropine sulfate
ophthalmic solution drops were purchased from Phoenix
Pharmaceutical (St. Joseph, Mo.). All other chemicals were reagent
grade or better.
[0073] Animals. Male Long-Evans rats (Harlan Sprague-Dawley,
Madison, Wis.), which weighed 250-350 g, were used throughout these
experiments. All animals were supplied food and water ad libitum
and maintained on a 12 hour light/dark schedule in a temperature-
and humidity-controlled environment. Animals were handled in
accordance with the Guide for the Care and Use of Laboratory
Animals as adopted and promulgated by the National Institutes of
Health.
[0074] Methanol-Intoxication Protocol. Animals were randomly
assigned to one of four treatment groups: (1) Untreated control,
(2) LED-treated control, (3) methanol-intoxicated and (4)
LED-treated methanol- intoxicated rats. Rats were placed in a
thermostatically controlled plexiglass chamber
(22.times.55.times.22 cm; maintained at 22-23.degree. C.) and
exposed to a mixture of N.sub.2O/O.sub.2 (1:1; flow rate 2
liters/min) for the duration of the experiment. N.sub.2O/O.sub.2
exposure produces a transient state of tetrahydrofolate deficiency
in the rat resulting in formate accumulation following methanol
administration (J. T. Eells, et al., supra, 2000). In the present
studies, methanol (25% w/v methanol in saline) was administered
(i.p.) to N.sub.2O/O.sub.2 treated rats at an initial dose of dose
4 g/kg, followed by supplemental doses of 1.5 g/kg at 24 and 48
hours. This methanol intoxication protocol has been shown to
produce a state of prolonged formic acidemia with formate
concentrations between 5-8 mM in methanol-intoxicated rats
resulting in visual dysfunction (M. T. Seme, et al., supra, 1999;
M. T. Seme, et al., supra, 2001). Moreover, similar concentrations
of blood formate over similar time periods have been shown to
produce ocular toxicity experimentally in monkeys and have been
associated with visual toxicity in human methanol intoxication (J.
T. Eells, supra, 1992; R. Kavet and K. Nauss, supra, 1990; S. O.
lngemansson, supra, 1983). Formate concentrations were determined
from tail vein blood samples by fluorometric analysis as previously
described (M. T. Seme, et al., supra, 1999; M. T. Seme, et al.,
supra, 2001; T.G. Murray, et al., 1991).
[0075] Light-Emitting Diode Treatment. GaAIAs light emitting diode
(LED) arrays of 670 nm wavelength (LED bandwidth 25-30 nm at 50%
power) were obtained from Quantum Devices, Inc. (Barneveld, Wis.).
Rats were placed in a plexiglass restraint device
(12.7.times.9.times.7.6 cm). The LED array was positioned directly
over the animal at a distance of 1 inch, exposing the entire body.
Treatment consisted of irradiation at 670 nm for 2 minutes and 24
seconds resulting in a power intensity of 28 mW/ cm.sup.2 and an
energy density of 4 joules/cm.sup.2 at 5, 25 and 50 hours after the
initial dose of methanol. These stimulation parameters (670 nm at
an energy density of 4 J/cm.sup.2) had been demonstrated to be
beneficial for wound healing, and to stimulate cellular
proliferation and cytochrome oxidase activity in cultured visual
neurons (H. T. Whelan, et al., supra, 2001; M. T. T. Wong-Riley, et
al., supra, 2001).
[0076] ERG Procedures and Analyses. ERG experiments were performed
as previously described (M. T. Seme, et al., supra, 1999; M. T.
Seme, et al., supra, 2001). The light stimulation apparatus
consisted of a three-beam optical system. All three beams were
derived from tungsten-halide lamps (50 W, 12 V). Beam intensity was
controlled by using neutral density step filters. ERG recordings
were differentially amplified and computer-averaged. The amplified
signal was processed through a two-stage active narrow bandpass
filter (the half voltage of this filter was 0.2 times the center
frequency). To ensure that any transients in the response that
occur at the onset of the stimulus pulses were not included in the
average, the initiation of signal averaging was delayed by a preset
number of stimulus cycles (typically a minimum of 20). The
resulting ERG is an extremely noise-free, single cycle, sinusoidal
waveform. The averaged responses were measured (peak-to-trough
amplitude) from a calibrated digital oscilloscope display.
[0077] Before ERG analysis, ophthalmoscopic examination confirmed
that all eyes were free of lenticular opacities or other gross
anomalies. Rats were anesthetized with thiobutabarbitol sodium salt
(100 mg/kg, i.p.), positioned in a Kopf stereotaxic apparatus and
placed on a heating pad to maintain core body temperature at
37.degree. C. Atropine sulfate (0.05 mg/kg, s.q.) was administered
to inhibit respiratory-tract secretions. The pupil of the eye to be
tested was dilated by topical application of 1% atropine sulfate.
Methylcellulose was topically applied as a lubricant and to enhance
electrical conduction. A circular silver, wire recording electrode
was positioned on the cornea, a reference electrode was placed
above the eye, and a ground electrode was placed on the tongue.
Recordings were obtained under ambient light conditions from cool
white fluorescent room lights approximately 100 cd/m.sup.2 at the
rat's eye. Flickering stimuli (light/dark ratio=0.25:0.75) were
presented. Responses to 60 successive flashes were averaged for
each stimulus condition. At each test wavelength, a minimum of four
stimulus intensities spaced at intervals of 0.3 log unit, were
presented. The stimulus intensity yielding a 5-.mu.V criterion
response was determined by extrapolating between the two intensity
points that bracketed the 5-.mu.V response for each animal. All
sensitivity measures were made in triplicate.
[0078] Two experimental protocols were used to evaluate retinal
function. (J. F. Rizzo, supra, 1995)
[0079] 15 Hz/510 nm ERG Response. ERGs were recorded in response to
a 15-Hz flickering light at a wavelength of 510 nm over a 3-log
unit range of light intensity. For these studies, the unattenuated
stimulus (log relative retinal illumination=0) had an irradiance of
25 .mu.W distributed over the 70.degree. patch of illuminated
retina. This can be calculated to produce retinal illumination
equivalent to about 10.sup.4 scotopic trolands. These recording
conditions disadvantage rods; however, since at least 97% of rat
photoreceptors are rods and ERGs are recorded at luminance
intensities ranging from 10.sup.1 to 10.sup.4 scotopic trolands, it
is likely that the responses to the 15 Hz/510 nm light are drawn
from both rods and medium wavelength cones (M-cones) (M. T. Seme,
et al., supra, 1999; M. T. Seme, et al., supra, 2001; D. A. Fox and
L. Katz, Vision Res. 32:249-255, 1992).
[0080] 25 Hz/UV ERG Response. UV-sensitive cone responses were
elicited by a 25-Hz flickering ultraviolet light (380-nm cut off)
in the presence of an intense chromatic adapting light (445 .mu.W)
which eliminated responses mediated by rods and M-cones (G. Jacobs,
et al., Nature 353:655-656, 1991). The 25-Hz/UV ERG responses were
recorded over a 1.5-log unit range of light intensity. For these
studies, the unattenuated stimulus (log relative retinal
illumination=0) had an irradiance of 25 .mu.W distributed over the
70.degree. patch of illuminated retina. This can be calculated to
produce retinal illumination equivalent to about 10.sup.4 scotopic
trolands in the rat eye.
[0081] Histopathologic Analysis. Retinal tissue was prepared for
histology as previously described (M. T. Seme, et al., supra, 1999;
M. T. Seme, et al., supra, 2001). Thick sections (1.mu.) for light
microscopy were stained with toluidine blue; thin sections for
electron microscopy were stained for uranyl acetate-lead citrate
(M. T. Seme, et al., supra, 1999; M. T. Seme, et al., supra,
2001).
[0082] Statistical Analysis. All values are expressed as
means.+-.SEM. A one-way ANOVA with Bonferroni's test was used to
determine whether any significant differences existed among groups
for blood formate concentrations. For ERG studies, a two-way ANOVA
was performed. In all cases, the minimum level of significance was
taken as P<0.05.
[0083] Results
[0084] Blood formate accumulation in methanol-intoxicated rats is
not altered by 670 nm LED treatment. Formic acid is the toxic
metabolite responsible for the retinal and optic nerve toxicity
produced in methanol intoxication (G. Martin-Amat, et al., supra,
1977; J. T. Eells, supra, 1992; J. T. Eells, et al., supra, 2000;
G. Martin-Amat, al., supra, 1978). Linear increases in blood
formate concentrations were observed in both methanol-intoxicated
and LED-treated methanol-intoxicated rats during the 72-hour
intoxication period (FIG. 1).
[0085] Referring to FIG. 1, photobiomodulation does not alter blood
formate concentrations in methanol-intoxicated rats. Blood formate
concentrations were determined before methanol administration and
at 24 hour intervals after methanol administration for 72 hours.
Shown are the mean values.+-.SEM from six rats in each experimental
group. Blood formate concentrations did not differ between the
methanol-intoxicated and LED-treated, methanol-intoxicated groups
(P>0.05).
[0086] In both treatment groups, blood formate concentrations
increased tenfold from endogenous concentrations of 0.5-0.6 mM
prior to methanol administration to nearly 6 mM following 72 hours
of intoxication. The rate of formate accumulation and blood formate
concentrations did not differ between the two treatment groups,
indicating that LED treatment did not alter methanol or formate
toxicokinetics. Similar increases in blood formate have been shown
to disrupt retinal function in methanol intoxicated rats (M. T.
Seme, et al., supra, 1999; M. T. Seme, et al., supra, 2001) and
have been associated with visual toxicity in human methanol
intoxication (J. T. Eells, supra, 1992; R. Kavet and K. Nauss,
supra, 1990).
[0087] Methanol-induced retinal dysfunction is attenuated by 670-nm
LED treatment. Following 72 hours of methanol intoxication, the
function of rods and M-cones was assessed by recording the retinal
response to a 15-Hz flickering light at wavelength of 510 nm (M. T.
Seme, et al., supra, 1999; M. T. Seme, et al., supra, 2001).
[0088] Referring to FIG. 2, photobiomodulation improves rod and
M-cone ERG response in methanol-intoxicated rats. Rod and M-cone
(15 Hz/510 nm) ERG analysis was performed after 72 hours of
methanol intoxication. Shown are the mean values.+-.SEM from six
rats in the untreated control, methanol-intoxicated, and
LED-treated, methanol-intoxicated experimental groups and four rats
from the LED control group. ERG responses in methanol-intoxicated
and LED-treated, methanol-intoxicated rats were significantly lower
than those measured in control rats (*, P<0.001). ERG responses
in LED-treated, methanol-intoxicated rats were significantly
greater than those measured in methanol-intoxicated rats (,
P<0.001).
[0089] In the untreated control group, 15-Hz/510-nm ERG amplitude
increased linearly over the 3-log unit range of retinal
illumination intensities, achieving a maximal amplitude of 65.+-.5
.mu.V at maximal retinal illumination (0 log relative retinal
illumination (LRRI) equivalent to 10.sup.4 scotopic trolands). A
similar ERG response profile was observed in LED-control animals.
In both control groups a consistent 5-.mu.V criterion threshold
response was obtained at -3.0.+-.0.1 LRRI. In agreement with our
previous studies, methanol intoxication produced a profound
decrease in retinal sensitivity to light coupled with an
attenuation of maximal ERG response amplitude (M. T. Seme, et al.,
supra, 1999; M. T. Seme, et al., supra, 2001). The light intensity
required to elicit a threshold (5 .mu.V) 15-Hz/510-nm ERG response
was increased by 0.6 log units to -2.4.+-.0.1 LRRI in
methanol-intoxicated rats relative to control animals. In addition,
the amplitudes of the flicker ERG responses were significantly
attenuated at all luminance intensities achieving a maximal
amplitude of 18.+-.5 .mu.V, approximately 28% of the maximum
control response. These changes are indicative of a severe deficit
in retinal function and are consistent with formate-induced
inhibition of photoreceptor oxidative metabolism (M. T. Seme, et
al., supra, 1999; M. T. Seme, et al., supra, 2001; G. Jacobs, et
al., supra, 1991; A. Koskelainen, et al, Vision Res. 34:983-994,
1994; O. Findl, et al., Invest. Ophthalmol. Visual Sci.
36:1019-1026, 1995). LED treatment significantly improved rod and
M-cone mediated ERG responses in methanol intoxicated rats. At
lower luminance intensities (<1.5 LRRI), LED treatment had no
effect on ERG response; however, at luminance intensities >1.5
LRRI, ERG responses were significantly greater in LED-treated rats
compared to methanol intoxicated animals. The maximal rod and
M-cone in LED-treated rats was 47.+-.8 .mu.V, 72% of the maximal
control response. These data are indicative of a partial recovery
of rod and M-cone function by LED photobiomodulation in
methanol-intoxicated rats.
[0090] The function of UV-sensitive cones was examined by recording
the retinal response to a 25-Hz flickering ultraviolet light
(380-nm cutoff) in the presence of an intense chromatic adapting
light. These conditions have been shown to isolate the UV-cone
response in the rat retina (G. Jacobs, et al., supra, 1991). The
effects of methanol intoxication and LED light treatment on UV-cone
ERG responses are shown in FIG. 3.
[0091] Referring to FIG. 3, photobiomodulation improves UV-cone ERG
response in methanol-intoxicated rats. UV-cone (25 Hz/380 nm) ERG
analysis was performed after 72 hours of methanol intoxication.
Shown are the mean values.+-.SEM from six rats in the control,
methanol-intoxicated, and LED-treated, methanol-intoxicated
experimental groups and four rats from the LED control group.
UV-cone ERG responses were recorded from the same animals in which
the rod and M-cone responses were recorded. ERG responses in
methanol-intoxicated and LED-treated, methanol-intoxicated rats
were significantly lower than those measured in control rats (*,
P<0.001). ERG responses in LED-treated, methanol-intoxicated
rats were significantly greater than those measured in
methanol-intoxicated rats (, P<0.05).
[0092] In untreated control animals the UV-cone-mediated ERG
amplitude increased linearly from a 5-.mu.V threshold value
(-1.4.+-.0.03 LRRI) to a maximal value of 56.+-.3 .mu.V over the
1.5-log unit range of retinal illumination used in these studies.
LED-treated control animals exhibited a similar ERG response
profile to that observed in untreated control animals. In
methanol-intoxicated rats, the UV-cone ERG response was profoundly
attenuated consistent with our previous studies (M. T. Seme, et
al., supra, 1999; M. T. Seme, et al., supra, 2001). The light
intensity required to elicit a 5-.mu.V response was increased by
0.5 log units to 0.9.+-.0.08 LRRI in intoxicated animals, and the
maximal response amplitude was reduced to 18.+-.6 .mu.V, 30% of the
maximum control response. Similar to what we observed in the rod
and M-cone ERG studies, LED treatment had no effect on UV-cone ERG
response at lower luminance intensities, but significantly improved
ERG response at higher luminance intensities. The maximal UV-cone
ERG response in LED-treated rats was 37.+-.7 .mu.V, 61% of the
control response indicative of a partial recovery of UV-cone
function by LED photobiomodulation.
[0093] Methanol-induced retinal histopathology is prevented by 670
nm LED treatment. The architecture of the retina in
methanol-intoxicated and LED-treated methanol intoxicated rats was
evaluated by light and electron microscopy. These studies focused
on the outer retina at the level of the photoreceptors based on our
previous findings of outer retinal pathology and photoreceptor
mitochondrial disruption following methanol intoxication (M. T.
Seme, et al., supra, 1999; M. T. Seme, et al., supra, 2001; T. G.
Murray, et al., supra, 1991). FIG. 4 illustrates outer retinal
morphology in representative untreated control (FIG. 4A), LED
control (FIG. 4B), methanol intoxicated (FIG. 4C), and LED-treated
methanol intoxicated (FIG. 4D) retinas.
[0094] Referring to FIG. 4, photobiomodulation protects retinal
morphology in methanol-intoxicated rats. Outer retinal morphology
in representative untreated control (A), LED control (B),
methanol-intoxicated (C), and LED-treated, methanol-intoxicated (D)
rats. Sections were taken from the posterior pole of the retina
within two disk diameters of the optic nerve in any direction.
(Toluidine blue, .times.450.) (A) rpe, retinal pigment epithelium;
os, photoreceptor outer segments; is, photoreceptor inner segments;
onl, outer nuclear layer; opl, outer plexiform layer; ipl, inner
plexiform layer. (B) The arrows indicate enlargement and swelling
of the photoreceptor inner segments, and the circles indicate the
fragmented appearance of photoreceptor nuclei. No histopathologic
changes were apparent at the light microscopic level in the LED
control or LED-treated, methanol-intoxicated groups.
[0095] Pronounced histopathologic changes were apparent in the
outer retina of methanol-intoxicated rats (FIG. 4C), including
evidence of retinal edema, swelling of photoreceptor inner
segments, and morphologic changes in photoreceptor nuclei. Retinal
edema was evidenced by the spacing between the photoreceptor inner
segments, and by the spacing of the nuclei in the outer nuclear
layer. Photoreceptor inner segments were profoundly swollen and
enlarged, and photoreceptor nuclei in the outer nuclear layer
appeared fragmented with irregularly stained chromatin. In
contrast, LED-treated methanol-intoxicated animals (FIG. 4D)
exhibited retinal morphology which was indistinguishable from
untreated control rats (FIG. 4A) and LED treated control rats (FIG.
4B). In these animals outer retinal morphology was characterized by
ordered photoreceptor inner segments with no evidence of
vacuolization or swelling and the outer nuclear layer was compact
with round and well-defined nuclei. The lack of retinal
histopathology in LED-treated methanol intoxicated rats provides
additional evidence of the retinoprotective actions of 670-nm LED
treatment.
[0096] The most obvious ultrastructural change observed in the
outer retina of methanol-intoxicated rats was swelling and
disruption of mitochondria in the inner segments of the
photoreceptors. Referring to FIG. 5, photobiomodulation protects
photoreceptor ultrastructure in methanol-intoxicated rats. Electron
micrographs of the rod inner segment region in representative
untreated control (A), LED control (B), methanol-intoxicated (C),
and LED-treated, methanol-intoxicated (D) rats. The arrows indicate
abnormal mitochondrial morphology in photoreceptor inner segments.
Photoreceptor mitochondria from LED control or LED-treated,
methanol- intoxicated rats exhibited normal morphology with
well-defined cristae, (Magnifications: .times.5,000).
[0097] Some mitochondria were swollen and contained expanded
cristae; other mitochondria were disrupted and showed no evidence
of cristae (FIG. 5C). In contrast, mitochondria in the
photoreceptor inner segments from LED-treated, methanol-intoxicated
rats (FIG. 5D) exhibited normal morphology with well-defined
cristae similar to inner segment mitochondrial morphology in
untreated control rats (FIG. 5A) and LED-treated control rats (FIG.
5B). The absence of mitochondrial damage in photoreceptors of
LED-treated methanol-intoxicated rats strongly supports our
hypothesis that 670-nm LED treatment preserved mitochondrial
function.
[0098] Discussion
[0099] Low-energy photon irradiation by light in the far-red to
near-IR spectral range 630-1000 nm) using low-energy lasers or LED
arrays has been found to modulate various biological processes in
cell culture and animal models (M. J. Conlan, et al., supra, 1996;
W. Yu, et al., supra, 1997; A. P. Sommer, et al., supra, 2001; T.
Karu, supra, 1999). This phenomenon of photobiomodulation has been
applied clinically in the treatment soft tissue injuries and to
accelerate wound healing (H. T. Whelan, et al., supra, 2001; M. J.
Conlan, et al., supra, 1996). The mechanism of photobiomodulation
by red to near-IR light at the cellular level has been ascribed to
the activation of mitochondrial respiratory chain components,
resulting in initiation of a signaling cascade which promotes
cellular proliferation and cytoprotecton (T. Karu, supra, 1999; N.
Grossman, et al., supra, 1998; M. T. T. Wong-Riley, et al., supra,
2001). A comparison of the action spectrum for cellular
proliferation after photoirradiation with the absorption spectrum
of potential photoacceptors lead Karu (T. Karu, supra, 1999) to
suggest that cytochrome oxidase is a primary photoreceptor of light
in the red to near-IR region of the spectrum.
[0100] Recent studies conducted in primary neuronal cultures by our
research group have shown that 670-nm LED photobiomodulation
reversed the reduction in cytochrome oxidase activity produced by
the blockade of voltage-dependent sodium channel function by
tetrodotoxin and up-regulated cytochrome oxidase activity in normal
neurons (M. T. T. Wong-Riley, et al., supra, 2001). The present
studies extended these investigations to an in vivo system to
determine if 670-nm LED photobiomodulation would improve retinal
function in an animal model of formate-induced mitochondrial
dysfunction. Results of this study demonstrate the therapeutic
benefit of photobiomodulation in the survival and functional
recovery of the retina in vivo after acute injury by the
mitochondrial toxin, formic acid generated in the course of
methanol intoxication. We provide in vivo evidence that three brief
post-methanol-intoxication treatments with 670-nm LED
photoirradiation promotes the recovery of retinal function in rod
and cone pathways and protects the retina from the histopathologic
changes induced by methanol-derived formate. These findings provide
a link between the actions of red to near-IR light on mitochondrial
oxidative metabolism in vitro and retinoprotection in vivo.
[0101] Low-energy laser irradiation has documented benefits in
promoting the healing of hypoxic, ischemic, and infected wounds (H.
T. Whelan, et al., supra, 2001; M. J. Conlan, et al., supra, 1996).
However, lasers have limitations in beam width, wavelength
capabilities, and size of wounds that can be treated (H. T. Whelan,
et al., supra, 2001). Heat generated from the laser light can
damage biological tissue, and the concentrated beam of laser light
may accidentally damage the eye. LED arrays were developed for
National Aeronautics and Space Administration manned space flight
experiments. In comparison to lasers, the patented LED technology
generates negligible amounts of heat, is clinically proven to be
safe, and has achieved non-significant risk status for human trials
by the Food and Drug Administration (H. T. Whelan, et al., supra,
2001). The wavelength, power, and energy parameters used in the
present study are based on their beneficial effects for wound
healing in humans (H. T. Whelan, et al., supra, 2001) and
stimulation of CO activity in cultured neuronal cells (M. T. T.
Wong-Riley, et al., supra, 2001).
[0102] The retinoprotective actions of 670-nm LED treatment in the
present study are consistent with the actions of formate as a
mitochondrial toxin and the actions of 670-nm light on cytochrome
oxidase activity. Formate has been shown to reversibly inhibit
cytochrome oxidase activity with an apparent inhibition constant
between 5 and 30 mM (P. Nicholls, supra, 1975; P. Nicholls, supra,
1976). Blood formate concentrations in methanol-intoxicated rats in
the present study fall within this range and retinal formate
concentrations closely parallel blood formate concentrations (J. T.
Eells, et al., supra, 2000). The functional and morphologic
alterations produced in the retina by methanol-derived formate are
indicative of formate-induced inhibition of photoreceptor
mitochondrial energy metabolism. Photoreceptors are the most
metabolically active cells in the body, and the energy required for
phototransduction is derived primarily from oxidative metabolism
(A. Ames, III, et al., J. Neurosci. 12:840-853, 1992; A. Ames, III,
Can. J. Physiol. Pharmacol. 70:S158-S164, 1992). The loss of
retinal sensitivity to light and attenuation of ERG response in
methanol-intoxicated rats are indicative of formate-induced
inhibition of photoreceptor oxidative energy metabolism and are
similar to the actions of other metabolic poisons in the retina (M.
T. Seme, et al., supra, 1999; A. Koskelainen, et al., supra, 1994;
O. Findl, et al., supra, 1995). The observed mitochondrial swelling
and disruption in the photoreceptor inner segments in
methanol-intoxicated rats are consistent with a disruption of ionic
homeostasis secondary to inhibition of cytochrome oxidase.
Moreover, similar morphologic alterations have been reported in the
retinas of patients with mitochondrial diseases that inhibit
electron transport (P. A. McKelvie, et al., J. Neurol. Sci.
102:51-60, 1991; L. M. Rapp, etal., Invest. Ophthalmol. Visual Sci.
31:1186-1190, 1990; P. Runge, et al., Br. J. Opththalmol.
70:782-796, 1986).
[0103] In the present study, the increase in ERG response and the
lack of damage to photoreceptor mitochondria in LED-treated,
methanol-intoxicated rats are indicative of a biostimulatory effect
of 670-nm light on photoreceptor bioenergetics. A growing body of
evidence suggests that cytochrome oxidase is a key photoacceptor of
light in the far red to near infrared spectral range (T. Karu,
supra, 1999; W. Yu, et al., supra, 1997; S. Passarella, et al.,
FEBS Lett. 175:95-99, 1984; D. Pastore, et al., Int. J. Radiat.
Biol. 76:863-870, 2000). Cytochrome oxidase is an integral membrane
protein which contains four redox active metal centers and has a
strong absorbance in the far-red to near-IR spectral range
detectable in vivo by near-IR spectroscopy (C. E. Cooper and R.
Springett, Philos. Trans. R. Soc. London B352:9-676, 1977; B.
Beauvoit, et al., Anal. Biochem. 226:167-174, 1995; B. Beauvoit, et
al., Biophys. J. 67:2501-2510, 1994). Moreover, 660-680 nm
irradiation has been shown to increase electron transfer in
purified cytochrome oxidase (D. Pastore, et al., supra, 2000),
increase mitochondrial respiration and ATP synthesis in isolated
mitochondria (S. Passarella, et al., supra, 1984), and to up
regulate cytochrome oxidase activity in cultured neuronal cells (M.
T. T. Wong-Riley, et al., supra, 2001). An up-regulation of retinal
cytochrome oxidase by LED treatment would effectively counteract
the inhibitory actions of formate on retinal oxidative metabolism,
thus improving retinal function. Although retinal function was
improved in LED-treated rats, it was not restored to control
response levels. At lower luminance intensities, LED treatment did
not improve the ERG response in methanol-intoxicated rats
suggesting that the rate of activation of some components of
phototransduction activation remained compromised by formate.
Because the rate of activation of phototransduction depends on an
adequate supply of GTP and ATP (G. Jacobs, et al., supra, 1991; A.
Koskelainen, et al., supra, 1994; O. Findl, et al., supra, 1995),
it is possible that the formate-induced metabolic inhibition is
only partly attenuated by our LED treatment protocol.
[0104] The prolonged effect of three brief LED treatments in
mediating the retinoprotective actions in methanol intoxication
suggests that 670-nm LED photostimulation induces a cascade of
signaling events initiated by the initial absorption of light by
cytochrome oxidase. These signaling events may include the
activation of immediate early genes, transcription factors,
cytochrome oxidase subunit gene expression, and a host of other
enzymes and pathways related to increased oxidative metabolism (T.
Karu, supra, 1999; M. T. T. Wong-Riley, et al., supra, 2001; C.
Zhang and M. Wong-Riley, Eur. J. Neurosci. 12:1013-1023, 2000). In
addition to increased oxidative metabolism, red to near-IR light
stimulation of mitochondrial electron transfer is also known to
increase the generation of reactive oxygen species (T. Karu, supra,
1999). These mitochondrially generated reactive oxygen species may
function as signaling molecules to provide communication between
mitochondria and the cytosol and nucleus and thus play an important
signaling role in the activation of retinoprotective processes
following LED treatment (S. Nemoto, et al., Mol. Cell. Biol.
20:7311-7318, 2000).
[0105] The results of this study demonstrate that
photobiomodulation with red to near-IR light augments recovery
pathways promoting neuronal viability and restoring neuronal
function following injury. Importantly, there was no evidence of
damage to the normal retina following 670-nm LED treatment. Based
on these findings, we propose that photobiomodulation represents an
innovative and novel therapeutic approach for the treatment of
retinal injury, as well as the treatment of retinal diseases
including age-related macular degeneration, glaucoma, diabetic
retinopathy and Leber's hereditary optic neuropathy.
EXAMPLE 2
Animal Model of Retinal Protection.
[0106] Methanol intoxication produces toxic injury to the retina
and optic nerve frequently resulting in blindness. The toxic
metabolite in methanol intoxication is formic acid, a mitochondrial
toxin known to inhibit the essential mitochondrial enzyme,
cytochrome oxidase. The Eells' laboratory has developed a rodent
model of methanol toxicity which replicates the metabolic and
retinotoxic manifestations of human methanol toxicity. This animal
model also manifests many features associated with retinal aging
and many clinically important retinal and optic nerve diseases and
thus serves as an excellent experimental model for the
investigation of treatments for retinal and optic nerve
disease.
[0107] Studies were undertaken to determine if exposure to
monochromatic 670 nm radiation from light-emitting diode (LED)
arrays would protect the retina against the toxic actions of
methanol-derived formic acid in this animal model of ocular
disease. Methanol-intoxicated and non-intoxicated control rats were
placed in a plexiglass restraint device (12.7.times.9.times.7.6
cm). The LED array was positioned directly over the animal at a
distance of 2.5 cm. Treatment consisted of irradiation at 670 nm
for 2 min and 24 sec resulting in a power intensity of 28 mW/
cm.sup.2 and an energy density of 4 joules/cm.sup.2. NIR-LED
treatments were administered 5, 25 and 50 hours after the initial
dose of methanol. These stimulation parameters (670 nm at an energy
density of 4 J/cm.sup.2 ) have been demonstrated to be beneficial
for wound healing, and to stimulate cellular proliferation and
cytochrome oxidase activity in cultured visual neurons.
[0108] The electroretinogram (ERG) which measures the response of
the retina to flickering light stimulation was used as a sensitive
and clinically relevant indicator of retinal function. NIR-LED
treated animals exhibited a dramatic improvement in retinal
function measured by the ERG (FIG. 6) and NIR-LED treatment also
protected the retina from the histopathologic damage induced by
methanol-derived formate. These findings provide a link between the
actions of monochromatic red to near infrared light on
mitochondrial oxidative metabolism in vitro and retinoprotection in
vivo.
EXAMPLE 3
Nonhuman Primate Model of Retinal Protection.
[0109] We have initiated studies of laser retinal injury in a
nonhuman primate model. To date, we have performed two experiments
using this animal model. In each experiment one monkey was lased
without LED treatment and one lased with LED treatment (670 nm, 4
J/cm.sup.2). A laser grid (128 spots delivered to the macula and
perimacula) was created in the central retina of right eye of each
animal. This grid consisted of grade I and II burns,
photocoagulating the photoreceptors and outer nuclear layer of the
retina. Multifocal ERG was performed to assess the functional state
of the retina. In the first experiment, the LED-treated monkey was
treated at 1, 24 ,72 and 96 hours post injury. ERG amplitude in
both LED treated and untreated monkeys was temporarily increased
shortly after laser injury and this increase was greater in the
LED-treated monkey. Assessment of the severity of the laser burn in
LED treated and untreated animal demonstrated a greater that 50%
improvement in the degree of retinal healing at 1 month post-laser
in the LED-treated monkey (FIG. 7). In addition, the thickness of
the retina measured at the fovea by optical coherence tomography
did not differ from the pre-laser thickness in the LED-treated
animal whereas it was 50% thinner in the untreated animal.
Importantly, LED treatment prevented the loss of cytochrome oxidase
staining (FIG. 8) in the lateral geniculate nucleus clearly showing
that the brain was responding to visual input from the "healed"
retina in the LED-treated animal much more effectively than in the
untreated animal.
[0110] In the second study, the LED-treated animal was treated once
per day for 11 days and mfERG recordings were recorded (FIG. 9).
Again, shortly after laser injury, the ERG amplitude was
temporarily increased in both LED treated and untreated animals.
However, in this experiment the increases were comparable. At 4
days post laser injury, the mfERG responses in LED treated and
untreated animals had decreased to pre-laser amplitudes. However,
by day 11 post laser, the mfERG response in the LED treated monkey
was more than 50% greater than that measured in the untreated
(sham) monkey. In both experiments, these preliminary findings are
indicative of improved retinal healing and visual cortical function
following LED treatment in laser injured primate model.
EXAMPLE 4
Effect of NIR-LED Treatment in Leber's Hereditary Optic
Neuropathy.
[0111] The effect of NIR-LED treatment was investigated in the
treatment of Leber's Hereditary Optic Neuropathy (LHON). LHON is a
disease caused by a mitochondrial mutation (the most common
mutation is in position 11778 of the mitochondrial genome) which
results in defective mitochondrial energy production and causes
blindness in early adulthood.
[0112] NIR-LED treatment was investigated in 6 affected (blind)
11778 LHON mutation carriers in Colatina, Brazil according to the
protocol approved by the Institutional Review Board of San Paolo
Federal University. Each subject exhibited a profound deficit in
central vision. Baseline values for NSE, Humphrey 60.degree. visual
fields and nerve fiber analysis were obtained prior to LED
treatment. LED treatment consisted of irradiation at 670 nm for 80
seconds delivered to each eye producing an estimated energy density
of 4 joules/cm.sup.2 at the optic nerve head. LED treatment was
administered once per day for 3 days using handheld LED arrays
(Quantum Devices, Barneveld, Wis.) positioned 2.5 cm from each
closed eye. Treatment response was assessed 1 day following the
third LED treatment (day 4) and again on day 10. Two of the
NIR-LED-treated subjects reported a transient improvement in color
vision and visual acuity lasting approximately one day. NSE
concentrations in these two subjects increased dramatically (from a
pre-exposure level of 0.9 .mu.g/L to 7.6 .mu.g/L in one subject and
2.2 .mu.g/L to 5.3 .mu.g/L in the other) in contrast to smaller
increases or decreases in NSE measured in the other four subjects.
Peripheral visual fields showed distinct improvement in 4 of the 6
patients by 10 days post treatment. No change was observed in nerve
fiber layer measurements. No detrimental effects of NIR-LED
treatment were reported by study subjects and no adverse effects
were observed in visual function tests. The findings of this pilot
study confirm and extend previous studies which have reported that
NIR-LED exposure at energy densities up to 300 joules/cm.sup.2
produces no detrimental effects on the retina and optic nerve. The
studies further demonstrate that NIR-LED treatment exerts a
beneficial effect in LHON.
* * * * *